| dc.description.abstract | With the growth of DNA nanotechnology, significant efforts have been made to expand the chemical functionality of DNA beyond the genetic code carrier. By leveraging the specificity of the interactions and modularity afforded, combined with easy bioconjugation of DNA, DNA molecules can be utilized to ‘sense’, ‘transduce’, ‘report’ and act as a scaffold for such functional elements. Over the last couple of decades, a new class of DNA molecules known as “aptamers” have been discovered which bind to their target with high specificity and selectivity. DNA aptamer-based detection usually employs ligand-induced structural changes or conformation stabilization that is
coupled to optical, electrochemical, or mass-based detection schemes. Among them, fluorescence-based aptamer biosensors provide a highly sensitive, background free optical sensing method. Of the numerous fluorescence-based approaches, Förster resonance energy transfer (FRET) and protein-induced fluorescence enhancement (PIFE) stand out because of their sensitivity at the nanometer scale, accurate quantification and ease of implementation. As a proof-of-concept, we employed two DNA aptamers (that bind to kanamycin and thrombin) to show that this approach can report on ligand binding without prior knowledge of structural changes in the aptamer. We show that the ratiometric FRET-based analysis can be implemented on a cheap custom-built smartphone setup that can detect kanamycin in a linear range of 50–500 nM with the limit of detection (LOD) of 28 nM. Similarly, the exquisite sensitivity of PIFE to protein binding near the aptamer dye can be employed to detect thrombin in the linear detection range of 0.25 pM–25 nM with a LOD of 8.9 pM. Our fluorescent aptasensors based simple detection scheme(s) perform comparably to known methods as a homogeneous ‘one-pot’ assay for thrombin and kanamycin in their ‘natural’ blood plasma and milk background, respectively.
Next, we explored (lipophilic) DNA reporters to understanding the lipid diffusion and 2D assembly in the lipid bilayer membranes. In a crowded but dynamic lipid cell membrane, diffusion of macromolecules deviates from Brownian motion and displays ‘anomalous’ diffusion. A common underlying physical mechanism: the presence of slower-moving obstacles, heterogeneous diffusion, transient binding, pinning sites, and compartmentalization have all been suggested as potential contributors to the anomaly in membrane diffusion. Here, we attempted to study the anomalous diffusion by controlling the polymer (PEG) labelled lipids content in the supported lipid bilayers (SLBs) using single-particle tracking of DNA reporters. By systematically varying the PEG content to induce domains in the SLBs, we are able to tune the nature and size of these local ‘domains’. Next, we extended this reporter system to study the room temperature self-assembly process of M13mp18 DNA nanostructures on the crowded bilayer membranes. | en_US |
